Mouse monoclonal antibodies can be relatively easily isolated by the widely used hybridoma technology (Kohler, G. and Milstein, C. Nature (1975) 256, 495–497). On the other hand, a similar technique for human hybridoma has yet to be widespread though it is expected to become so. Furthermore, there is a need for antibodies to human antigens in clinical applications, and therefore the generation of mouse monoclonal antibodies is indispensable for the development of antibody pharmaceuticals.
In fact, a number of monoclonal antibodies have been isolated against tumor cells and viruses, and have been studied in clinical applications. It has been revealed, however, that mouse antibodies, which are a foreign substances to humans, induce HAMA (human anti-mouse antibody) due to the potent antigenicity, and that it is extremely unsuitable for clinical applications because of such problems as a weak activity of inducing ADCC (Schroff, R. W., Cancer Res. (1985) 45, 879–885; Shawler, D. L., et al, J. Immunol. (1985) 135, 1530–1535).
In order to solve this problem, chimeric antibody was created (Neuberger, M. S. et al., Nature (1984) 312, 604–608; Boulianne, G. L. et al., Nature (1984) 312, 643–646). Chimeric antibody is made by linking a variable region of a mouse antibody to a constant region of a human antibody, i.e. in chimeric antibody the constant region of the mouse antibody which is responsible for a particularly potent antigenicity has been replaced with a human counterpart. This is expected to enable a physiological binding with a human Fc receptor and to induce Fc-mediated functions. In fact, marked decreases in antigenicity has been reported in a clinical study using chimeric antibodies (LoBuglio, A. F. et al., Proc. Natl. Acad. Sci. U.S.A. (1989) 86, 4220–4224). However, trouble-causing cases were reported that developed HAMA against mouse variable regions (LoBuglio, A. F. et al., Proc. Natl. Acad. Sci. U.S.A. (1989) 86, 4220–4224).
Accordingly, methods have been developed, though more complicated, for making a humanized antibody which is closer to a human antibody. This is a technique of reconstructing the antigen binding site of a mouse antibody on a human antibody (Jones, P. T. et al., Nature (1986) 321, 5225–525; Verhoeyen, M. et al., Scinece (1988) 239, 1534–1536; Riechmann, L. et al., Nature (1988) 332,323–327)). Thus, a variable region of an antibody, for both of the H chain and the L chain, comprises four framework regions (FRS) and three complementarity determining regions (CDRs) sandwiched between them.
It is known that CDR is mainly responsible for the formation of antigen binding sites and some amino acid residues on the FR are involved therein either directly or indirectly. Since the basic structures of antibodies are similar to each other, it was thought possible to graft an antigen binding site of an antibody to another antibody. The research group led by G. Winter has, in fact, successfully grafted CDRs of a mouse anti-rhizobium antibody to a human antibody (CDR-grafting) thereby obtaining a humanized antibody having a rhizobium binding activity (Jones, P. T. et al., Nature (1986) 321, 522–525).
In some cases, however, humanization by CDR-grafting alone does not provide humanized antibody that has an antigen binding activity similar to the original mouse antibody. Accordingly, as described above, attempts have been made to replace some FR amino acid residues. FR amino acid residues to be replaced are involved in the maintenance of the structure of amino acid residues that constitute the basic structure of an antibody molecule (canonical structure; Chothia, C. et al., Nature (1989) 342, 877–883; Chothia, C. and Lesk, A. M. J. Molec. Biol. (1987) 196, 901–917) or CDRs, or directly interact with antigen molecules.
In fact, amino acid substitution on the FR has been made for most of the humanized antibody, wherein artificial FR sequences that do not naturally occur are formed. At times, too many amino acid substitutions have been made, which makes doubtful the original meaning of CDR-grafting for minimizing the antigenicity of mouse antibody (Queen, C. et al., Proc. Natl. Acad. Sci. U.S.A. (1989) 86, 10029–10033; Co, M. S. et al., Proc. Natl. Acad. Sci. U.S.A. (1991) 88, 2869–2873).
A solution to this problem is to devise methods of selecting human FRS. Thus, the number of FR amino acid residues to be replaced depends on the homology between the FRs of the human antibody selected for CDR-grafting and the FRs of the original mouse antibody. Accordingly, human FRs having a high homology with mouse FRs are usually selected so as to minimize the degree of substitution. However, in many cases even the FRs of humanized antibody thus obtained have amino acid sequences that do not occur naturally, which may present the problem of antigenicity. Thus, there is a need for the technology of constructing humanized antibody that can solve the above problems, have lower probability of inducing antigenicity, and have higher safety.